Nano‐Brake Halts Mitochondrial Dysfunction Cascade to Alleviate Neuropathology and Rescue Alzheimer's Cognitive Deficits

Abstract Mitochondrial dysfunction has been recognized as the key pathogenesis of most neurodegenerative diseases including Alzheimer's disease (AD). The dysregulation of mitochondrial calcium ion (Ca2+) homeostasis and the mitochondrial permeability transition pore (mPTP), is a critical upstream signaling pathway that contributes to the mitochondrial dysfunction cascade in AD pathogenesis. Herein, a “two‐hit braking” therapeutic strategy to synergistically halt mitochondrial Ca2+ overload and mPTP opening to put the mitochondrial dysfunction cascade on a brake is proposed. To achieve this goal, magnesium ion (Mg2+), a natural Ca2+ antagonist, and siRNA to the central mPTP regulator cyclophilin D (CypD), are co‐encapsulated into the designed nano‐brake; A matrix metalloproteinase 9 (MMP9) activatable cell‐penetrating peptide (MAP) is anchored on the surface of nano‐brake to overcome the blood‐brain barrier (BBB) and realize targeted delivery to the mitochondrial dysfunction cells of the brain. Nano‐brake treatment efficiently halts the mitochondrial dysfunction cascade in the cerebrovascular endothelial cells, neurons, and microglia and powerfully alleviates AD neuropathology and rescues cognitive deficits. These findings collectively demonstrate the potential of advanced design of nanotherapeutics to halt the key upstream signaling pathways of mitochondrial dysfunction to provide a powerful strategy for AD modifying therapy.


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siRNA core with different magnesium ion and phosphate ion ratios. Scale bar: 50 nm.    siRNA concentration) to test the toxicity of this carrier) for 24 h, respectively. Then, the cell viability was measured by CCK8 assay, n=3-4, Data represent the mean ± SD. Figure S6. Nano-brake showed good storage stability. Number mean (A), zeta potential (B), and PDI (C) of Nano-brake after stored at 4°C for 0, 1, 4, 7, 14 days. (D), (E), Nano-brake protects siRNA from degradation. After stored at 4°C for 0, 1, 4, 7, 14 days, the remaining siRNA content in Nano-brake was determined. Data represent the mean ± SD. n=3.   Mg-CypD-LNC (41.3 μg/kg siRNA) via the tail vein of 6-month-old 5xFAD mice, the distribution of the DiI-Mg-CypD-LNC in the brain. Two-photon imaging images of DiI-Mg-CypD-LNC distributed in the cerebral vessels of 5xFAD mice. Scale bar, 40 μm. Figure S10. The brain entry efficiency of DiI-Nano-brake increased in 5xFAD mice.
Six month-old 5xFAD mice and littermate WT mice were injected with DiR fluorescent-labeled Nano-brake and Mg-CypD-LNC (82.5 μg/kg siRNA) via the tail vein, respectively. n=3-4. The statistical assay of DiR fluorescent intensity of the brain in Fig. 2E was analyzed by IVIS Spectrum/CT imaging system. Data represent the mean ± SEM.

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Figure S11. DiR-Nano-brake showed similar distribution profiles in the peripheral organs of 5xFAD mice with Mg-CypD-LNC. Six-month-old 5xFAD mice and littermate WT mice were injected with DiR-labeled Nano-brake and Mg-CypD-LNC (82.5 μg/kg siRNA) via the tail vein, respectively. A. The fluorescent images of the organs were taken via an IVIS Spectrum/CT imaging system. B. Quantification of DiR fluorescence intensity in heart, liver, spleen, lung, and kidney of 5xFAD mice. n=4.        Video S1. After administration of DiI labeled Nano-brake (41.3 μg/kg siRNA) via the tail vein of 6-month-old 5xFAD mice. Two-photon imaging video of Nano-brake distributed on the cerebral vessels of 5xFAD mice from 40 min to 70 min. The white arrow showed higher adhesion along and permeation across the cerebral vessels. Scale bar, 40 μm.

Experimental Section
Animals. 5xFAD mice were provided by the Jackson Laboratory (Bar Harbor, ME USA) and bred in the specific pathogen-free animal facility (SPF). The animals were raised in the SPF animal facility with free access to water and food at 22 ± 2°C and a light- Preparation of Nano-brake. Magnesium phosphate core was loaded with CypD siRNA (Mg-siRNA core) using a reverse water-in-oil microemulsion method, referring to our previous calcium phosphate core preparation method [1] . As shown in Figure 1A, 50 μL of 50 μM siRNA and 300 μL of 0.625 M MgCl2 were dispersed in 10 mL Igepal CO-520 / cyclohexane (7/3 V/V) solution to form a well dispersed microemulsion. Three 16 / 23 hundred microliters of 12.5 mM Na2HPO4 and 50 μL of 50 μM siRNA were added to 10 mL Igepal CO-520 / cyclohexane solution to prepare the phosphate part. DOPA (20 mM) chloroform solution (100 μL) was dropped into the above phosphate phase. The above two Igepal CO-520 / cyclohexane solutions were mixed and stirred for 40 min.
Then, 20 mL ethanol was added for emulsion breaking. The mixture was centrifuged at 12,000 g for 20 min, thus the surfactant of this mixture was removed carefully. After two thorough washes with ethanol, the pellets were dissolved in 2 mL chloroform and Characterization of Nano-brake. The nanocarriers' particle size and zeta potential were evaluated using a Zeta-sizer Nano-ZS90 system. Nano-brake were negative stained with 1.5% sodium phosphotungstate solution and characterized by TEM (JEOL, JEM-1400, Japan).
To evaluate the siRNA encapsulation efficiency of Mg-siRNA-LNC, cy3-siRNA was used as the fluorescent indicator. For the analysis, Mg-cy3-siRNA-LNC was dissolved in pH 7.8 Tris lysis buffer containing 2 mM EDTA and 0.05% Trixton X-100. They were then incubated at 65°C for 10 min to release the entrapped siRNA before being measured by the fluorescence microplate reader.

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The Mg 2+ level in the Nano-brake was quantitatively measured by Magnesium Assay Kit (Nanjing Jiangcheng Bioengineering Institute, China) based on the manufacturer's protocols.
They were then analyzed by electrophoresis using 2% agarose gel to monitor the remaining siRNA content.
The storage stability of Nano-brake. After stored at 4°C for 0, 1, 4, 7, 14 days, the size, zeta potential and PDI of Nano-brake were determined by Zeta-sizer Nano-ZS90 system. The siRNA in Nano-brake was analyzed by electrophoresis using agarose gel to test the remaining siRNA content.

Cell culture and cellular uptake of DiI-Nano-brake. bEnd.3 cells (Shanghai Type
Culture Collection of the Chinese Academy of Sciences, China.) were seeded in a 96well plate in DMEM medium containing 10% FBS, 100 μg/mL streptomycin, 100 units/mL penicillin and 1% non-essential amino acids at the density of 5×10 3 cells/well in a 37°C, 5% CO2 humidified incubator to 80% confluency. The cells were incubated with DiI-Nano-brake (at 5 μg/mL DMPC) in DMEM for 3 h at 37°C. After rinsing with PBS, the cells were fixed with a 4% formaldehyde solution. Following nuclei staining with Hoechst, the above cells were quantitatively analyzed via an HCS instrument (Thermo Scientific Cellomics, Thermo, USA) to evaluate the cellular uptake of DiI-Nano-brake according to the previous method [2] .

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The toxicity of Nano-Brake was assessed by CCK8 assay. The toxicity of Nano-brake

Detection of d9-DMPC nano-brake in the brain.
The deuterium isotope labeled d9nano-brake, or d9-Mg-CypD-LNC in the brain of mice was quantified by LC-MS/MS according to our previous method [3,4] . Briefly, d9-Nano-brake or d9-Mg-CypD-LNC was prepared using the same method by replacing 1 mg DMPC with 1 mg d9-DMPC during the LNC preparation process. Six months old 5xFAD and WT littermates mice were administered by caudal vein at a dose of 82.5 μg/kg siRNA and grouped as follows: A) 5xFAD + d9-Nano-brake; B) 5xFAD + d9-Mg-CypD-LNC; C) WT + d9-Nanobrake. The brain homogenates were extracted using methyl tert butyl ether, and 13:0 The proportion of administered dose per gram (%ID/g) was used to express the remaining amount of Nano-brake in the brain: Western blot. For cell, bEnd.3 cells were cultured in a 6-well plate at the density of 2.5 × 10 5 cells/well. Following culturing for 24 h, the cells were incubated with DMEM control, Mg-CypD-LNC (pre-incubating with MMP9 protein), Nano-brake and Nanobrake (pre-incubating with MMP9 protein), respectively, for 48 h at a concentration of 100 nM siRNA. And then, CypD expression was evaluated by western blot as described previously [5] . For western blot brain tissue sample preparation, the brain tissue and RIPA lysis solution (containing 1 mM PMSF) were added to the grinding tube with a weight-to-volume ratio of 1:3, and then grinding magnetic beads were added. After that, the tubes were placed in the tissue grinding homogenizer and fully cracked for 3 min to acquire the brain homogenate. The 20-40 μg of cell or brain tissue lysates were To visualize Nano-brake binding to cerebral vessels, in vivo multi-photon imaging was applied to observe cerebral vessels referring to our previous method [6] . Briefly, ketamine (100 mg/kg) and midazolam (5 mg/kg) were intraperitoneally administered to the mice to maintain general anesthesia. Then, a cranial window was opened in the parietal bone, and thin grass was carefully placed and fixed with dental cement. DiI- After that, the cells were washed twice with PBS, fixed in 2.5% glutaraldehyde, and then stained with OsO4 as described before [7] . Finally, the ultra-thin section was analyzed by a Hitachi 7600 electron microscope. Immunofluorescence and immunohistochemical analysis. Immunofluorescence analyses were performed based on our previous method [2] . The mice were euthanized as above mentioned and were perfused with 0.1 M PB and 4% formaldehyde solution for fixing brain tissue. Afterward, the brain was further preserved in 4% formaldehyde solution for 36 h, embedded in paraffin, and sectioned at a thickness of 4 μm. The brain slices were then immersed in citric acid for 15 min, and incubated with 3% H2O2 to quench the endogenous tissue peroxidase. carried out based on the previously described [2] . The training procedure lasted for four days, and the mice were trained 4 times each day. If the mice got the platform within 90 s, they were left to stay there for 10 s. Otherwise, they were led to the platform and remained for 30 s to bear in mind the platform location. On the probe trial day, let the mice swim in the pool without the platform. All analysis was recorded and analyzed by a tracking system (Shanghai Jiliang Software Technology, China).

Enzyme
Novel object recognition (NOR) test. The NOR test was finished based on our previous method [6] . The NOR test equipment contains an acrylic cube box (35 cm × 35 cm) with a white bottom. The test procedure is divided into three phases: habituation, familiarization, and test phase. On habituating day, each mouse was carefully placed into an empty box and given five minutes to explore the field. The area was cleaned with alcohol to clear the odor. On the day of training, two identical objects were located on the third point of the diagonal line of the box bottom. A mouse was placed in the center of the box and allowed to explore freely for 5 min. On the day of testing, one of the objects was substituted with a new one. The mice were placed in the box one by one and stayed for 5 min to recognize the two different objects, and their exploration track was recorded and analyzed by the tracking system. Y maze. Y maze device is made of a black medical organic board with three arms of the same size and shape at 30 cm*8 cm*15 cm, and the angle between arms is 120°.
The end of each wall is pasted with colorful figures with different shapes as a visual mark. Gently place the mouse into the middle area where the three arms meet and allow it to explore freely. Record the sequence of mice entering each arm within 8 min.
Alternation is defined as consecutively entering three arms, such as 1, 2, 3; 1, 3, 2; 3, 2, 1 or 2, 1, 3. As long as the mouse enters three different arms continuously in a row, it can be regarded as an alternation.
The alternation= ℎ -2 Statistical analysis. In the same experiment, the age and sex of mice were kept in the same conditions and randomly allocated to different experimental groups. GraphPad Prism 7.0 software was applied for the statistical analyses. The data represent the mean ± SEM or mean ± SD, as described in the figure legend. Differences between the two groups were examined using Student's t-test, and ANOVA with Tukey's multiple comparison tests for 3 or more groups. Significant differences were considered if p < 0.05.